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Sequencing

Targeted Testing

Turnaround Time

The great majority of tests are completed within 18 days.

Clinical Sensitivity

This test is predicted to detect pathogenic variants in <0.1% of individuals with intellectual disability (ID). In this context, it is important to note that although pathological variants over 800 genes have been identified in individuals with ID, a genetic diagnosis is still lacking in most cases due to extreme clinical and genetic heterogeneity of the condition (Vissers et al. 2016). Analytical sensitivity should be high because all pathogenic variants reported within this gene to date are detectable by sequencing.

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Clinical Features

Intellectual Disability (ID) is a heterogeneous group of neurodevelopmental disorders, characterized by congenital limitation in intellectual functioning (intelligence quotient, IQ, below 70). ID is diagnosed in ~1–3% of the population before 18 years of age (American Association of Intellectual and Developmental Disabilities, AAIDD).

Baraitser-Winter syndrome 1 (BRWS1, syndromic ID) is a complex developmental disorder that is usually characterized by variable ID with epilepsy, craniofacial anomaly (facial and nasal dysmorphism, hypertelorism, oral cleft, etc.), congenital ptosis and ocular coloboma (Baraitser and Winter 1988). Some affected individuals can also present with postnatal short stature, microcephaly, skeletal abnormality and sensoneuronal deafness (Fryns and Aftimos 2000; Rivière et al. 2012; Verloes et al. 2015). Pathogenic variants in the human ‘actin beta’ gene (ACTB) have been reported in several cases of BRWS1. Interestingly, in vitro studies on the lymphoblastoid cell lines of these patients did not show any difference in the protein levels compared to the controls, however, cytoskeletal changes and abnormal accumulations of F-actin rich filopodia-like protrusions have been observed in patients, which is likely to impact the cell morphology, motility, and other actin-related functions (Rivière et al. 2012).

Genetics

Pathogenic variants in ACTB (mostly de novo) result in Baraitser-Winter syndrome 1 (BRWS1) (Rivière et al. 2012). ACTB maps to chromosome 7p22.1 and consists of 5 coding exons that encode a 375 amino acid polypeptide. Actin beta (ACTB) is one of the six vital cytoskeletal actins and is a ubiquitous, highly-conserved globular protein. Since the actin family of proteins are implicated in nearly all cellular processes (including cell motility), Actb-null mouse are embryonically lethal (Shawlot et al. 1988). To date, only missense heterozygous variants have been reported in ACTB, although there are a few reports of gross deletions/duplications events that include ACTB and additional genes. The disease transmission pattern is consistent with autosomal dominant inheritance. Interestingly, several observations support a dominant-negative or gain-of-function mechanism for the disease-causing variants in ACTB gene (Rivière et al. 2012).

Testing Strategy

The testing strategy involves PCR amplification followed by bidirectional Sanger sequencing of the full coding exons plus ~20 bp of adjacent noncoding sequences. We will also sequence any single exon (Test #100) in family members of patients with a known mutation or to confirm research results.

Indications for Test

This test is primarily implicated for patients with syndromic intellectual disabilities who are negative for any kind of cytogenetic abnormalities, copy number variations, Fragile-X syndrome, and also for the family members of the patients who have ACTB pathogenic variants. Prenatal diagnosis is possible, if the genetic diagnosis has been firmly established in an affected family member.

TEST METHODS

Bi-Directional Sanger Sequencing

Test Procedure

Nomenclature for sequence variants was from the Human Genome Variation Society (http://www.hgvs.org). As required, DNA is extracted from the patient specimen. PCR is used to amplify the indicated exons plus additional flanking non-coding sequence. After cleaning of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit. Products are resolved by electrophoresis on an ABI 3730xl capillary sequencer. In most cases, sequencing is performed in both forward and reverse directions; in some cases, sequencing is performed twice in either the forward or reverse directions. In nearly all cases, the full coding region of each exon as well as 20 bases of non-coding DNA flanking the exon are sequenced.

Analytical Validity

As of March 2016, we compared 17.37 Mb of Sanger DNA sequence generated at PreventionGenetics to NextGen sequence generated in other labs. We detected only 4 errors in our Sanger sequences, and these were all due to allele dropout during PCR. For Proficiency Testing, both external and internal, in the 12 years of our lab operation we have Sanger sequenced roughly 8,800 PCR amplicons. Only one error has been identified, and this was due to sequence analysis error.

Our Sanger sequencing is capable of detecting virtually all nucleotide substitutions within the PCR amplicons. Similarly, we detect essentially all heterozygous or homozygous deletions within the amplicons. Homozygous deletions which overlap one or more PCR primer annealing sites are detectable as PCR failure. Heterozygous deletions which overlap one or more PCR primer annealing sites are usually not detected (see Analytical Limitations). All heterozygous insertions within the amplicons up to about 100 nucleotides in length appear to be detectable. Larger heterozygous insertions may not be detected. All homozygous insertions within the amplicons up to about 300 nucleotides in length appear to be detectable. Larger homozygous insertions may masquerade as homozygous deletions (PCR failure).

Analytical Limitations

In exons where our sequencing did not reveal any variation between the two alleles, we cannot be certain that we were able to PCR amplify both of the patient’s alleles. Occasionally, a patient may carry an allele which does not amplify, due for example to a deletion or a large insertion. In these cases, the report contains no information about the second allele.

Similarly, our sequencing tests have almost no power to detect duplications, triplications, etc. of the gene sequences.

In most cases, only the indicated exons and roughly 20 bp of flanking non-coding sequence on each side are analyzed. Test reports contain little or no information about other portions of the gene, including many regulatory regions.

In nearly all cases, we are unable to determine the phase of sequence variants. In particular, when we find two likely causative mutations for recessive disorders, we cannot be certain that the mutations are on different alleles.

Our ability to detect minor sequence variants, due for example to somatic mosaicism is limited. Sequence variants that are present in less than 50% of the patient’s nucleated cells may not be detected.

Runs of mononucleotide repeats (eg (A)n or (T)n) with n >8 in the reference sequence are generally not analyzed because of strand slippage during PCR and cycle sequencing.

Unless otherwise indicated, the sequence data that we report are based on DNA isolated from a specific tissue (usually leukocytes). Test reports contain no information about gene sequences in other tissues.

Ship blood tubes at room temperature in an insulated container. Do not freeze blood.

During hot weather, include a frozen ice pack in the shipping container.
Place a paper towel or other thin material between the ice pack and the blood tube.

In cold weather, include an unfrozen ice pack in the shipping container as insulation.

At room temperature, blood specimen is stable for up to 48 hours.

If refrigerated, blood specimen is stable for up to one week.

Label the tube with the patient name, date of birth and/or ID number.

DNA

(Delivery accepted Monday - Saturday)

Send in screw cap tube at least 5 µg -10 µg of purified DNA at a concentration of at least 20 µg/ml for NGS and Sanger tests and at least 5 µg of purified DNA at a concentration of at least 100 µg/ml for gene-centric aCGH, MLPA, and CMA tests, minimum 2 µg for limited specimens.

For requests requiring more than one test, send an additional 5 µg DNA per test ordered when possible.

DNA may be shipped at room temperature.

Label the tube with the composition of the solute, DNA concentration as well as the patient’s name, date of birth, and/or ID number.

We only accept genomic DNA for testing. We do NOT accept products of whole genome amplification reactions or other amplification reactions.

CELL CULTURE

(Delivery preferred Monday - Thursday)

PreventionGenetics should be notified in advance of arrival of a cell culture.

Culture and send at least two T25 flasks of confluent cells.

Some panels may require additional flasks (dependent on size of genes, amount of Sanger sequencing required, etc.). Multiple test requests may also require additional flasks. Please contact us for details.

Send specimens in insulated, shatterproof container overnight.

Cell cultures may be shipped at room temperature or refrigerated.

Label the flasks with the patient name, date of birth, and/or ID number.

We strongly recommend maintaining a local back-up culture. We do not culture cells.